Intact Protein Research Articles

Using in vivo intact structure for system-wide quantitative analysis of changes in proteins

Mass spectrometry-based methods can provide a global expression profile and structural readout of proteins in complex systems. Preserving the in vivo conformation of proteins in their innate state is challenging during proteomic experiments. Here, we introduce a whole animal in vivo protein footprinting method using perfusion of reagents to add dimethyl labels to exposed lysine residues on intact proteins which provides information about protein conformation. When this approach is used to measure dynamic structural changes during Alzheimer’s disease (AD) progression in a mouse model, we detect 433 proteins that undergo structural changes attributed to AD, independent of aging, across 7 tissues. We identify structural changes of co-expressed proteins and link the communities of these proteins to their biological functions. Our findings show that structural alterations of proteins precede changes in expression, thereby demonstrating the value of in vivo protein conformation measurement. Our method represents a strategy for untangling mechanisms of proteostasis dysfunction caused by protein misfolding. In vivo whole-animal footprinting should have broad applicability for discovering conformational changes in systemic diseases and for the design of therapeutic interventions.

Prokaryotic expression of wheat TaABI5-D3 gene and polyclonal antibody preparation

Abscisic acid insensitive 5 (ABI5) is a basic leucine zipper transcription factor regulating ABA-mediated seed germination, and TaABI5 is closely related to the pre-harvest sprouting in wheat. Studies have shown that TaABI5-D3, one of multiple copies of TaABI5 gene, encodes the intact TaABI5 protein. In this study, we constructed the prokaryotic expression vector pET-28a-TaABI5-D3 for expression of TaABI5-D3 in Escherichia coli and obtained the purified recombinant protein His-TaABI5-D3. This protein existed in the form of inclusion bodies, with the best expression induced by incubation with 0.6 mmol/L IPTG at 16 ℃, 150 r/min overnight (for 12 h). Subsequently, the purified His-TaABI5-D3 was injected into Balb/C mice for the preparation of polyclonal antibodies. Western blotting analysis indicated that the polyclonal antibody had relatively high specificity, laying foundations for clarification of the function of TaABI5 protein in wheat.

Influence of different sample preparation approaches on proteoform identification by top-down proteomics.

Top-down proteomics using mass spectrometry facilitates the identification of intact proteoforms, that is, all molecular forms of proteins. Multiple past advances have lead to the development of numerous sample preparation workflows. Here we systematically investigated the influence of different sample preparation steps on proteoform and protein identifications, including cell lysis, reduction and alkylation, proteoform enrichment, purification and fractionation. We found that all steps in sample preparation influence the subset of proteoforms identified (for example, their number, confidence, physicochemical properties and artificially generated modifications). The various sample preparation strategies resulted in complementary identifications, substantially increasing the proteome coverage. Overall, we identified 13,975 proteoforms from 2,720 proteins of human Caco-2 cells. The results presented can serve as suggestions for designing and adapting top-down proteomics sample preparation strategies to particular research questions. Moreover, we expect that the sampling bias and modifications identified at the intact protein level will also be useful in improving bottom-up proteomics approaches.

Citrullination Accompanies the Development of Carotid Atherosclerotic Plaques.

Citrullination represents a post-translational modification primarily mediated by peptidylarginine deiminase (PADI) 2 and 4 and resulting in the conversion of positively charged peptidylarginine to neutrally charged peptidylcitrulline. Molecular consequences of citrullination include the generation of neoepitopes which provoke the production of autoantibodies implicated in the development of autoimmune diseases. As citrullination initiates, promotes, and is enhanced by aseptic inflammation which plays a pivotal role in atherosclerosis, we proposed that citrullination might accompany the development of atherosclerotic vascular disease. To investigate features and patterns of citrullination in atherosclerotic plaques. We collected carotid atherosclerotic plaques (n = 14) and adjacent arterial segments (n = 14) which were pairwise excised during the carotid endarterectomy. The tissues were examined employing proteomic profiling (ultra-high performance liquid chromatography-tandem mass spectrometry analysis), haematoxylin and eosin staining, Western blotting and immunofluorescence staining for peptidylcitrulline, PADI2, and PADI4, and gene expression analysis. To better explore the mechanisms of citrullination in the neointima, we have also stained excised plaques for the extracellular vesicle markers (CD9 and CD81) and assessed co-localisation of PADI2 (a citrullination marker) with CD81 (an extracellular vesicle marker). In order to study the systemic response to citrullination in an atherosclerotic vascular disease setting, we measured the level of anti-citrullinated protein antibodies in the serum of patients with ischaemic stroke and healthy volunteers. Proteomic profiling found 213 plaque-specific and 111 intact arteria-specific proteins, as well as 46 proteins and 13 proteins which have been respectively upregulated or downregulated in plaques as compared with the adjacent intact segments. Among the top 20 upregulated proteins were atherogenic apolipoprotein B-100, iron-associated protein haptoglobin, and matrix metal-loproteinase-9, together indicating the advanced stage of plaque progression. In comparison with the intact arterial segments, plaques demonstrated protein signatures of innate immune response and oxidative stress, suggesting aseptic inflammation as a driver of atherosclerotic vascular disease. Both peptidylcitrulline and PADI2 have been abundant in the neointima but negligible in tunica media; further, the levels of peptidylcitrulline, PADI2, and PADI4 were elevated in plaque lysates in comparison with those from adjacent arterial segments (p = 0.025, 0.025, and 0.010, respectively). Notably, PADI2 and peptidylcitrulline were co-localised with the cells in the neointima and a considerable proportion of PADI2 was co-localised with CD81-positive extracellular vesicles (p = 0.003). Albeit citrullinated histone H3 and myeloperoxidase showed higher signal in the neointima than in tunica media (p = 0.048 and 0.023, respectively), we did not observe any signs of neutrophil extracellular traps (e.g., unwound chromatin or co-localisation of citrullinated histone H3 with neutrophil elastase) in the plaque tissue. Serum anti-citrullinated protein antibodies were not elevated in patients with ischaemic stroke (p = 0.71), suggesting that vascular citrullination likely does not trigger a generalised immune response. The development of carotid atherosclerosis is associated with citrullination, although it represents a local rather than systemic phenomenon in this clinical scenario.

Immunocapture mass spectrometry: macroscopic history, recent trends and future prospects.

Literature on immunological pretreatment and mass spectrometry was searched in the Web of Science database. The collected data were utilized for bibliometric analysis and literature review to identify trends in this field. Immunological pretreatment initially began in chemical toxicology using gas chromatography mass spectrometry and research interests later shifted to biologics pharmacokinetics, biomarkers, anti-drug antibodies and diagnosis. The key advantage of using mass spectrometry was the ability to distinguish molecular subtypes. Future research interests are expected to focus on a broader range of analyte types (such as intact protein mass spectrometry) and methodological improvements, including better protein digestion and microfluidics, aiming for faster, less labor-intensive and more reliable analysis through multiplexing and automation.

Conformational Characterization of Peptides and Proteins by 193 nm Ultraviolet Photodissociation in the Collision Cell of a Trapped Ion Mobility Spectrometry-Time-of-Flight Mass Spectrometer.

Ultraviolet photodissociation (UVPD) has been shown to be a versatile ion activation strategy for the characterization of peptides and intact proteins among other classes of biological molecules. Combining the high-performance mass spectrometry (MS/MS) capabilities of UVPD with the high-resolution separation of trapped ion mobility spectrometry (TIMS) presents an opportunity for enhanced structural elucidation of biological molecules. In the present work, we integrate a 193 nm excimer laser in a TIMS-time-of-flight (TIMS-TOF) mass spectrometer for UVPD in the collision cell and use it for the analysis of several mass-mobility-selected species of ubiquitin and myoglobin. The resultant data displayed differences in fragmentation that could be correlated with changes in protein conformation. Additionally, this mobility-resolved UVPD strategy was applied to collision-induced unfolded ions of ubiquitin to follow changes in fragmentation patterns relating to the extent of protein unfolding. This platform and methodology offer new opportunities for exploring how conformational variations are manifested in the fragmentation patterns of gas-phase ions.

Direct recognition of an intact foreign protein by an αβ T cell receptor

αβ T cell receptors (αβTCRs) co-recognise antigens when bound to Major Histocompatibility Complex (MHC) or MHC class I-like molecules. Additionally, some αβTCRs can bind non-MHC molecules, but how much intact antigen reactivities are achieved remains unknown. Here, we identify an αβ T cell clone that directly recognises the intact foreign protein, R-phycoerythrin (PE), a multimeric (αβ)6γ protein complex. This direct αβTCR–PE interaction occurs in an MHC-independent manner, yet triggers T cell activation and bound PE with an affinity comparable to αβTCR–peptide–MHC interactions. The crystal structure reveals how six αβTCR molecules simultaneously engage the PE hexamer, mediated by the complementarity-determining regions (CDRs) of the αβTCR. Here, the αβTCR mainly binds to two α-helices of the globin fold in the PE α-subunit, which is analogous to the antigen-binding platform of the MHC molecule. Using retrogenic mice expressing this TCR, we show that it supports intrathymic T cell development, maturation, and exit into the periphery as mature CD4/CD8 double negative (DN) T cells with TCR-mediated functional capacity. Accordingly, we show how an αβTCR can recognise an intact foreign protein in an antibody-like manner.

Biofunctionalized dissolvable hydrogel microbeads enable efficient characterization of native protein complexes

The characterization of protein complex is vital for unraveling biological mechanisms in various life processes. Despite advancements in biophysical tools, the capture of non-covalent complexes and deciphering of their biochemical composition continue to present challenges for low-input samples. Here we introduce SNAP-MS, a Stationary-phase-dissolvable Native Affinity Purification and Mass Spectrometric characterization strategy. It allows for highly efficient purification and characterization from inputs at the pico-mole level. SNAP-MS replaces traditional elution with matrix dissolving during the recovery of captured targets, enabling the use of high-affinity bait-target pairs and eliminates interstitial voids. The purified intact protein complexes are compatible with native MS, which provides structural information including stoichiometry, topology, and distribution of proteoforms, size variants and interaction states. An algorithm utilizes the bait as a charge remover and mass corrector significantly enhances the accuracy of analyzing heterogeneously glycosylated complexes. With a sample-to-data time as brief as 2 hours, SNAP-MS demonstrates considerable versatility in characterizing native complexes from biological samples, including blood samples.

The crucial quality marker of Panax ginseng: Glycosylated modified ribonuclease-like storage protein

Panax ginseng C.A.Mey is a famous natural herbal medicine worldwide. Mountain-cultivated ginseng (MCG) and garden-cultivated ginseng (GCG) are two types of Panax ginseng. There is a significant difference in economic benefits between MCG and GCG, which can always lead to problems such as adulteration and substitution of MCG with lower-priced alternatives. We explored the quality marker of ginseng at the intact protein level and established a foundation for the quality control of ginseng. Cellulose nanocrystal assisted sample preparation combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) equipped with a high mass detector was performed to analyze intact proteins in ginseng. The results revealed that the ribonuclease-like storage protein is the most abundant protein in MCG and GCG. Meanwhile, the molecular weight of the ribonuclease-like storage protein showed great difference between different ginseng species, which is 26.2 kDa in MCG and 24.2 kDa in GCG. The ribonuclease-like storage protein glycosylation modification difference provides data support for the differentiation between MCG and GCG. This study showed that glycosylated modified ribonuclease-like storage protein can be a crucial quality marker of ginseng, facilitating the rapid distinction between MCG and GCG.

Proteomic analysis of the human amniotic mesenchymal stromal cell secretome by integrated approaches via filter-aided sample preparation

The immunomodulatory, anti-inflammatory and regenerative properties of the human amniotic mesenchymal stromal cells (hAMSCs) secretome are acknowledged but the understanding of the specific bioactive components remains incomplete. To address these limitations, the present investigation aimed to profile the proteins and peptides content of the hAMSC secretome through sample pretreatment and fractionation on 10 kDa molecular cut-off FASP (Filter Aided Sample Preparation) device and LC-MS analysis. The filter retained protein fraction underwent trypsin digestion, while the unretained was collected unchanged for intact small proteins and peptides analysis. This combined approach (C-FASP) collects in a single step two complementary fractions, advantageously saving sample volume and time of analysis. The bottom-up analysis of the C-FASP proteins fraction >10 kDa confirmed our previous findings, establishing a set of proteins consistently characterizing the hAMSC secretome. The analysis of the fraction <10 kDa, never been investigated to our knowledge, identified peptide fragments of thymosin beta 4 and beta 10, collagen alpha 1 chains I and III, alpha-enolase, and glyceraldehyde-3-phosphate dehydrogenase, involved in wound healing, anti-inflammatory response, tissue repair and regeneration, key biological activities of the secretome. C-FASP provided a comprehensive molecular profile of the hAMSC secretome offering new insights for enhanced therapeutic applications in regenerative medicine. SignificanceIn this investigation we originally present the comprehensive proteomic investigation of the human amniotic mesenchymal stromal cell secretome by combining the analysis of the proteome and of the peptidome following sample pretreatment and fractionation by Filter Aided Sample Preparation (FASP) with 10 kDa molecular cut-off in coupling with LC-MS analysis. The proteome fraction retained by FASP filter was analyzed after enzymatic digestion, while the unretained fraction, below 10 kDa molecular mass, was analyzed unchanged in its intact form. This dual approach provides novel insights, previously unexplored, into the molecular components potentially responsible for the immunomodulatory and anti-inflammatory properties of the hAMSC secretome. These findings could significantly enhance the therapeutic potential of hAMSCs in regenerative medicine.

Mass Spectrometry-based proteomics for the masses: Peptide and protein identification in the Hunt laboratory during the 2000’s

There has been a rapid increase in the number of individuals utilizing mass spectrometry (MS)-based proteomics to study complex biological systems and questions since the start of the 2000's. Building off the advancements in ionization and liquid chromatography scientists continued to push towards technology that would enable in-depth analysis of biological specimen. Donald F Hunt and the Hunt laboratory were major contributors to this effort with their work on improving upon existing Fourier Transform MS, development of electron transfer dissociation, and continued work on ion-ion reactions to improve intact protein analysis. Collaboration with other instrumentation laboratories and instrument companies led to the sharing of technology and eventual commercialization providing greater access. Additionally, the Hunt laboratory spread the gospel of mass spectrometry-based proteomics through collaborations that lasted decades with other scientists who were experts in immunology, cellular signaling, epigenetics, and other fascinating fields. This article attempts to highlight the many contributions of Don and the Hunt laboratory to peptide and protein identification since the year 2000.

Effect of In Vitro Digestion on Bioactive Peptides Related to Immune and Gut Health in Intact Cow's Milk and Hydrolyzed Protein-Based Infant Formulas.

Background/Objectives: Human milk is the optimal source of nutrition and protection against infection for infants. If breastfeeding is not possible, standard and hydrolyzed infant formulas (IF) are an alternative. Extensively hydrolyzed IFs (eHFs) contain bioactive peptides, but their activities have rarely been evaluated. The aim of this study was to characterize and compare the bioactive peptide profiles of different eHFs and standard IFs before and after in vitro digestion. Methods: Two forms, liquid and powder, of intact protein formula (iPF) and eHF were subjected to in vitro gastrointestinal digestion, mimicking a young infant's gut (age 0-4 months) and an older infant's gut (>6 months). Bioactive peptides of in vitro digested and undigested formulas were analysed with Liquid Chromatography-Mass Spectrometry (LC-MS). Results: In all samples, a variety of peptides with potential bioactive properties were found. Immuno-regulatory peptides, followed by antimicrobial and antioxidative peptides were most frequent, as were peptides promoting wound healing, increasing mucin secretion, regulating cholesterol metabolism, and preventing bacterial infection. Peptides typically found in yoghurt and colostrum were identified in some formula samples. Conclusions: The high amounts of bioactive peptides with various properties in eHFs and iPFs indicate a possible contribution to infection protection, healthy gut microbiomes, and immunological development of infants. eHFs showed similar compositions of bioactive peptides to iPFs, with intermittently increased peptide variety and quantity.

Design and Implementation of a Proof-of-Concept Robotic-Microfluidic Interface to Bridge Spatial Comprehensive Three-Dimensional Liquid Chromatography with Mass Spectrometry.

A proof-of-concept system is presented for the hyphenation of spatial comprehensive three-dimensional liquid chromatography (3D-LC) to mass spectrometry (MS) detection via a robotic-microfluidic interface. A three-dimensional fractal microflow distributor, incorporating 16 parallel RP monolithic capillary columns arranged in a 4 × 4 configuration, was connected to an X-Y-Z robotic system. This setup facilitated the deposition of successive arrays of microdroplets onto an MS target plate. To minimize carryover during droplet deposition, a strategy was implemented in which the distance between the target plate and the capillary was gradually increased during the deposition process. System-level variation in travel time and subsequent flow rates across parallel columns was assessed and translated in retention alignment based on injection of a protein standard. The successful separation of intact proteins was demonstrated through a parallel 4 × 4 column configuration, applying MALDI-MS detection after microdroplet spotting on an MS target plate. Furthermore, the discussion encompasses high-throughput MS imaging detection within the framework of spatial 3D-LC.

Comparative analysis of allosteric rearrangements in FtsZ protein structure induced by benzamide and 4-hydroxycoumarine compounds

Aim. To reveal allosteric rearrangements of FtsZ molecules arising under the influence of benzamide compounds and 4-hydroxycoumarin derivatives. To discover the key molecular mechanisms predetermining the effect of the specified compounds on the cell division in bacteria. Methods. Comparative analysis of FtsZ protein structures and their complexes with ligands. Application of structural bioinformatics software for molecular visualization, measurement of interatomic distances and approximation of intramolecular shifts based on RMSD indicators. Results. Revealed conformational changes in FtsZ protein molecules, induced by allosteric effectors: 4-hydroxycoumarin – 4HC and benzamide – 9PC (PC-190723). Allosteric deformations and their consequences for intact FtsZ protein molecules, there GTPase domains, H7 helixes and C-terminal domains were studied. Conclusions. It was clarified that the binding of benzamides causes more significant shifts in the structure of the FtsZ protein monomer, its C-terminal domain, and H7 helix. At the same time, 4-hydroxycoumarins deform the structure of the GTPase domain almost twofold effectively. Both classes of compounds prosses allosteric action through unique mechanisms that are largely realized through deformations and displacements of the H7 helix. Despite the fact that these compounds demonstrate different allosteric mechanisms of action, their final effect can be summarized to destructions in GTP pocket, protofilament interfaces and the general geometry of molecule.

Activating pyruvate kinase improves red blood cell integrity by reducing band 3 tyrosine phosphorylation

AbstractIn a phase 1 study (NCT04000165), we established proof of concept for activating pyruvate kinase (PK) in sickle cell disease (SCD) as a viable antisickling therapy. AG-348 (mitapivat), a PK activator, increased adenosine triphosphate (ATP) and decreased 2,3-diphosphoglycerate levels while patients were on treatment, in line with the mechanism of the drug. We noted that the increased hemoglobin (Hb) persisted for 4 weeks after stopping AG-348 until the end of study (EOS). Here, we investigated the pathways modulated by activating PK that may contribute to the improved red blood cell (RBC) survival after AG-348 cessation. We evaluated frozen whole blood samples taken at multiple time points from patients in the phase 1 study, from which RBC ghosts were isolated and analyzed by western blotting for tyrosine phosphorylation of band 3 (Tyr-p-bd3), ankyrin-1, and intact (active) protein tyrosine phosphatase 1B (PTP1B) levels. We observed a significant dose-dependent decrease in mean Tyr-p-bd3 from baseline in the patients, accompanied by an increase in the levels of membrane-associated ankyrin-1 and intact PTP1B, all of which returned to near baseline by EOS. Because PTP1B is cleaved (inactivated) by intracellular Ca2+-dependent calpain, we next measured the effect of AG-348 on ATP production and calpain activity and the plasma membrane Ca2+ ATPase pump–mediated efflux kinetics in HbAA and HbSS erythrocytes. AG-348 treatment increased ATP levels, decreased calpain activity, and increased Ca2+ efflux. Altogether, our data indicate that ATP increase is a key mechanism underlying the increase in hemoglobin levels upon PK activation in SCD. This trial was registered at www.clinicaltrials.gov as #NCT04000165.

Multi-pass, single-molecule nanopore reading of long protein strands

The ability to sequence single protein molecules in their native, full-length form would enable a more comprehensive understanding of proteomic diversity. Current technologies, however, are limited in achieving this goal1,2. Here, we establish a method for the long-range, single-molecule reading of intact protein strands on a commercial nanopore sensor array. By using the ClpX unfoldase to ratchet proteins through a CsgG nanopore3,4, we provide single-molecule evidence that ClpX translocates substrates in two-residue steps. This mechanism achieves sensitivity to single amino acids on synthetic protein strands hundreds of amino acids in length, enabling the sequencing of combinations of single-amino-acid substitutions and the mapping of post-translational modifications, such as phosphorylation. To enhance classification accuracy further, we demonstrate the ability to reread individual protein molecules multiple times, and we explore the potential for highly accurate protein barcode sequencing. Furthermore, we develop a biophysical model that can simulate raw nanopore signals a priori on the basis of residue volume and charge, enhancing the interpretation of raw signal data. Finally, we apply these methods to examine full-length, folded protein domains for complete end-to-end analysis. These results provide proof of concept for a platform that has the potential to identify and characterize full-length proteoforms at single-molecule resolution.

Human milk vs. Infant formula digestive fate: In vitro dynamic digestion and in vivo mini-piglet models lead to similar conclusions

Infant formula (IF), the only nutritionally adequate substitute for human milk (HM), still needs to be improved to be more biomimetic with HM, including in terms of digestive fate. The latter can be explored using different digestion models. The present study aimed to compare IF and HM digestion using in vivo (mini-piglet) and in vitro (dynamic system, DIDGI®) models. Fresh mature HM was collected and compared with a standard bovine IF. In vivo, 18 Yucatan mini-piglets (24-day-old) received HM or IF and were euthanized 30 min after the last meal. The entire digestive content was collected from the stomach to the colon. In vitro, the same meals were fed to an in vitro dynamic digestion model simulating the term infant at four weeks of age. Digesta were sampled regularly in the gastric and intestinal compartments. Structure (confocal microscopy and laser light scattering) and proteolysis (SDS-PAGE for residual intact proteins, OPA for hydrolysis degree, LC-MS/MS for peptides) were investigated along digestion. The digesta microstructure differed between HM and IF in a similar way between in vitro and in vivo digestion. In vitro gastric proteolysis of caseins and α-lactalbumin was significantly slower for HM than for IF, such as for the early intestinal proteolysis degree. In vitro bioaccessibility of free AAs explained only 30 % of the true ileal digestibility of AAs. Peptide mapping of caseins differed between HM and IF along their digestion. The relative peptide mapping data over six proteins from HM and IF were highly correlated between in vitro and in vivo digestion, particularly at 80 and 120 min of in vitro gastric digestion vs. in vivo stomach data and at 20 and 40 min of in vitro intestinal digestion vs. in vivo proximal jejunum data (r = 0.7–0.9, p < 0.0001, n = 1604). 40 to 50 % of the bioactive peptides identified in vivo were also found in vitro, with a good correlation of their abundances (r = 0.5, p < 0.0001, n = 61). Overall, in vitro and in vivo digestion were in good agreement, both indicating a different digestive fate for HM and IF.

Qualitative and quantitative characterization of elastin-like polypeptides combining low-PH/low-temperature bottom-up and intact protein liquid chromatography mass spectrometric analysis.

Elastin-like polypeptides (ELPs) are elastic and thermoresponsive biopolymers composed of VPGXG repeats (X can be any amino acid except proline), used in biomedical applications, for example, tissue engineering and drug delivery. As different variants of ELP are mostly produced fermentatively, there is a need for the development of analysis methods that allow for absolute protein quantification in both complex matrices and purified samples and MW determination of the final products. ELPs were intracellularly expressed in Escherichia coli quantified after cell lysis and enzymatic digestion using a proline-specific protease ProAlanase (Promega) at acidic conditions. Resulting peptides were separated by liquid chromatography, and mass spectrometry analysis was conducted by electrospray ionization high-resolution mass spectrometry using an Orbitrap mass spectrometer. The addition of a stable isotopically labeled internal standard enabled quantification in complex matrices. Prior to intact mass analysis, ELPs were purified from fermentation broth by inverse temperature cycling. Intact protein analysis was performed using reversed-phase liquid chromatography, and mass spectrometry analysis was conducted by electrospray ionization high-resolution mass spectrometry using a time-of-flight mass spectrometer. Absolute quantification of ELPs was achieved by utilizing ELP-specific properties, that is, proline-rich, soluble at low pH and low temperature. The repetitive nature of ELPs allows for sensitivity increase and use of higher dilution factors to minimize the matrix effects. Despite the lack of amino acids with charged side chains (Arg, His, Lys, Asp, and Glu) in ELP, we demonstrated successful intact protein analysis using reversed-phase LC coupled to electrospray ionization TOF MS. Moreover, truncated protein forms could be chromatographically separated and characterized as well as N-terminal modifications. Both methods combined enabled quantitative and qualitative characterization of fermentatively produced ELPs.

Abstract P429: Stronger Association of Angiotensinogen with Mortality than Renin or Lactate in Critical Illness

Introduction: Sepsis and septic shock remain global healthcare issues associated with high mortality rates affecting millions every year despite best care efforts. Activation of the renin-angiotensin-system (RAS) is an early event in sepsis, and renin levels may more strongly associate with mortality than serum lactate, a widely accepted index of patient status. We postulated that reduced levels of Angiotensinogen (AGT) may reflect a dysfunctional RAS driven by high renin levels but an attenuated expression of Ang II to adequately support tissue perfusion and blood pressure. This study compared the association with mortality of AGT to serum lactate and active renin in a subset of the VICTAS (Vitamin C, Thiamine, and Steroids in Sepsis) cohort (N=103) at baseline (day 0) prior to treatment. Methods: Serum levels of intact AGT and active renin protein were quantified by separate ELISAs. Biomarker discrimination for all-cause 30-day mortality was compared by receiver operating characteristic (ROC) curves generated by logistic regression models adjusted for age, sex, sequential organ failure assessment (SOFA) score, systolic blood pressure, and in-hospital vasopressor support. Association of serum AGT, renin, and lactate with mortality was assessed by Kaplan-Meier curves with log rank test and hazard ratios derived from Cox regression. Results: Median [interquartile range] serum levels for AGT were: 114 [78.2-205.4] nM; renin: 4.9 [2.2-13.6] pM; and lactate: 2.6 [1.7-3.8] mM. ROC curves revealed better discrimination of AGT for mortality than either active renin or lactate. The area under the curve (AUC) of AGT (0.82, 95% CI: 0.70, 0.93) was greater than lactate (AUC=0.68, 95% CI: 0.57, 0.80; p=0.022). Kaplan-Meier curves and log rank test also revealed that AGT levels below 114 nM associated with reduced survival. Renin levels greater than 4.9 pM were also associated with patient mortality albeit less than that for AGT while higher lactate levels (&gt;2.6 mM) were not associated with mortality in this cohort. Conclusion: AGT levels more strongly associated with 30-day mortality than renin or lactate in the VICTAS cohort. Reduced AGT may reflect increased consumption by high renin coupled with reduced expression by the liver ultimately impairing Ang II-AT 1 R tone in critically ill patients. The prospective assessment of circulating AGT may facilitate more precise therapeutic approaches treatment to restore a dysfunctional RAS and improve overall mortality in sepsis.

Automated Assignment of 15N And 13C Enrichment Levels in Doubly-Labeled Proteins.

Uniform enrichment of 15N and 13C in proteins is commonly employed for 2D heteronuclear NMR measurements of the three-dimensional protein structure. Achieving a high degree of enrichment of both elements is important for obtaining high quality data. Uniform labeling of proteins and glycoproteins expressed in higher organisms (yeast or mammalian cell lines) is more challenging than expression in Escherichia coli, a prokaryote that grows on simple, chemically defined media but does not provide appropriate eukaryotic modifications. It is difficult to achieve complete incorporation of both heavy isotopes, and quality control measures are important for quantitating the level of their enrichment. Mass spectrometry measurements of the isotopic distribution of the intact protein or its proteolytic fragments provide the means to assess the enrichment level. A mass accuracy of 1 ppm or better is shown to be required to distinguish the correct combination of 13C and 15N enrichment due to subtle shifts in peak centroids with differences in the underlying, but unresolved, isotopic fine structure. A simple computer program was developed to optimize the fitting of experimental isotope patterns to statistically derived distributions. This method can determine the isotopic abundance from isotope patterns and isotopologue masses in conventional MS data for peptides, intact proteins, and glycans. For this purpose, MATLAB's isotope simulator, isotopicdist, has been modified to permit the variation of 15N and 13C enrichment levels and to perform a two-dimensional grid search of enrichment levels of both isotopes. We also incorporated an alternate isotope simulator, js-emass, into MATLAB for use in the same fitting program. Herein we benchmark this technique on natural abundance ubiquitin and uniformly [15N,13C]-labeled ubiquitin at both the intact and peptide level, outline considerations for data quality and mass accuracy, and report several improvements we have made to the previously reported analysis of the [15N,13C]-enriched human IgG Fc domain, a glycoprotein that has been expressed in Saccharomyces cerevisiae.